Treatment and Diagnostic Methods for Fibrosis Related Disorders

ABSTRACT

The present invention relates to the ability of SAP to suppress fibrocytes. It also relates to the ability of IL-12, laminin-1, cross-linked IgG and IgG aggregates to suppress fibrocytes. Methods and compositions for suppressing fibrocytes using these proteins are provided. These methods are useful in a variety of applications including treatment and prevention of fibrosing diseases such as scleroderma, pulmonary fibrosis and asthma. Finally, the invention includes assays for detecting the ability of various agents to modulate differentiation into fibrocytes. Such assays may also be used to diagnose scleroderma, pulmonary fibrosis, or other fibrosing diseases.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.11/535,636 filed Sep. 27, 2006, published as US 2007/0065368, which is acontinuation-in-part of U.S. application Ser. No. 11/158,966 filed Jun.22, 2005, published as US 2005/0238620 filed Jun. 22, 2005, which is acontinuation-in-part under 35 U.S.C. §120 of PCT patent applicationserial number PCT/US2003/040957, filed Dec. 22, 2003 and titled “Methodsand Conditions for Suppressing Fibrocyte Differentiation”, published inEnglish as WO 2004/058292 on Jul. 15, 2004; which claims priority to thefollowing: U.S. Provisional Patent Applications: U.S. 60/436,046, filedDec. 23, 2002; U.S. 60/436,027, filed Dec. 23, 2002; U.S. 60/515,776,filed Oct. 30, 2003; U.S. 60/519,467, filed Nov. 12, 2003; and U.S.60/525,175 filed Nov. 26, 2003. Pertinent parts of all aboveapplications are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to the ability of SAP, IL-12, Laminin-1,IgG aggregates, and/or cross-linked IgG to suppress fibrocytes.Accordingly, it may include compositions and methods for suppressingfibrocytes. These compositions and methods may be useful in a variety ofapplications, for example, those in which decreased fibrocyte formationis beneficial, such as treatment of fibrosing diseases and asthma. Theinvention may additionally include methods for detecting problems infibrocyte proliferation or differentiation, or for SAP, IL-12,Laminin-1, IgG aggregates, and/or cross-linked IgG to inhibit thisproliferation or differentiation. These problems may be correlated witha disease or may be drug-induced.

BACKGROUND Fibrocytes

Inflammation is the coordinated response to tissue injury or infection.The initiating events are mediated by local release of chemotacticfactors, platelet activation, and initiations of the coagulation andcomplement pathways. These events stimulate the local endothelium,promoting the extravasation of neutrophils and monocytes. The secondphase of inflammation is characterized by the influx into the tissue ofcells of the adaptive immune system, including lymphocytes. Thesubsequent resolution phase, when apoptosis of the excess leukocytes andengulfment by tissue macrophages takes place, is also characterized byrepair of tissue damage by stromal cells, such as fibroblasts.

In chronic inflammation, the resolution of inflammatory lesions isdisordered, with the maintenance of inflammatory cells, fibroblasthyperplasia, and eventual tissue destruction. The mechanisms that leadto these events are complex, but include enhanced recruitment, survivaland retention of cells and impaired emigration.

The source of fibroblasts responsible for repair of wound lesions or inother fibrotic responses is controversial. The conventional hypothesissuggests that local quiescent fibroblasts migrate into the affectedarea, produce extracellular matrix proteins, and promote woundcontraction or fibrosis. An alternative hypothesis is that circulatingfibroblast precursors (called fibrocytes) present within the bloodmigrate to the sites of injury or fibrosis, where they differentiate andmediate tissue repair and other fibrotic responses.

Fibrocytes are known to differentiate from a CD14+ peripheral bloodmonocyte precursor population. Fibrocytes express markers of bothhematopoietic cells (CD45, MHC class II, CD34) and stromal cells(collagen types I and III and fibronectin). Mature fibrocytes rapidlyenter sites of tissue injury where they secrete inflammatory cytokines.Fibrocytes are also capable of secreting extracellular matrix proteins,other cytokines and pro-angiogenic molecules, which may result infibrosis.

Control of fibrocyte differentiation is likely to be important in thecontrol of many diseases and processes. Fibrocytes are associated with avariety of processes and diseases including scleroderma, keloidscarring, rheumatoid arthritis, lupus, nephrogenic fibrosing dermopathy,and idiopathic pulmonary fibrosis. They play a role in the formation offibrotic lesions after Schistosoma japonicum infection in mice and arealso implicated in fibrosis associated with autoimmune diseases.Fibrocytes have also been implicated in pathogenic fibrosis, fibrosisassociated with radiation damage, Lyme disease and pulmonary fibrosis.CD34+ fibrocytes have also been associated with stromal remodeling inpancreatitis and stromal fibrosis, whereas lack of such fibrocytes isassociated with pancreatic tumors and adenocarcinomas. Fibrosisadditionally occurs in asthma patients and possibly other pulmonarydiseases such as chronic obstructive pulmonary disease when fibrocytesundergo further differentiation into myofibroblasts.

Fibrocytes may also play a role in a variety of conditions, likely evensome in which fibrocyte formation is not currently known. Someadditional conditions may include congestive heart failure, otherpost-ischemic conditions, post-surgical scarring including abdominaladhesions, corneal refraction surgery, and wide angle glaucomatrabeculectomy. Fibrocytes are also implicated in liver fibrosis andcirrhosis. See Tatiana Kisseleva et al, Bone Marrow-Derived FibrocytesParticipate in Pathogenesis of Liver Fibrosis, 45 Journal of Hepatology429-438 (September 2006); see also F. P. Russo et al, The Bone MarrowFunctionality Contributes to Liver Fibrosis, 130(6) Gastroenterology1807-21 (May 2006). Fibrocytes are important in the formation of tumors,particularly stromal tissue in tumors. Recent evidence also suggeststhat fibrocytes may further differentiate into adipocytes and thus playa role in obesity.

Serum Amyloid P

SAP, a member of the pentraxin family of proteins that includeC-reactive protein (CRP), is secreted by the liver and circulates in theblood as stable pentamers. The exact role of SAP is still unclear,although it appears to play a role in both the initiation and resolutionphases of the immune response. SAP binds to sugar residues on thesurface of bacteria leading to their opsonisation and engulfment. SAPalso binds to free DNA and chromatin generated by apoptotic cells at theresolution of an immune response, thus preventing a secondaryinflammatory response. Molecules bound by SAP are removed fromextracellular areas due to the ability of SAP to bind to all threeclassical Fcγ receptors (FcγR), with a preference for FcγRI (CD64) andFcγRII (CD32). After receptor binding, SAP and any attached molecule arelikely engulfed by the cell.

FcγR are necessary for the binding of IgG to a wide variety ofhematopoietic cells. Peripheral blood monocytes express both CD64 andCD32 (a subpopulation of monocytes express CD16), whereas tissuemacrophages express all three classical FcγR. Clustering of FcγR onmonocytes by IgG, either bound to pathogens or as part of an immunecomplex, initiates a wide variety of biochemical events. The initialevents following receptor aggregation include the activation of a seriesof src kinase proteins. In monocytes, these include lyn, hck and fgr,which phosphorylate tyrosine residues on the ITAM motif of the FcR-γchain associated with FcγRI and FcγRIII, the ITAM motif within thecytoplasmic domain of FcγRIIa or the ITAM motif with the cytoplasmicdomain of FcγRIIb. Phosphorylated ITAMs lead to the binding of a secondset of src kinases, including syk. Syk has been shown to be vital forphagocytosis of IgG-coated particles. However, the wide distribution ofsyk in non-hematopoietic cells and the evidence that syk is involved inboth integrin and G-protein coupled receptor signaling, indicates thatthis molecule has many functions.

Both SAP and CRP augment phagocytosis and bind to Fcγ receptors on avariety of cells. CRP binds with a high affinity to FcγRII (CD32), alower affinity to FcγRI (CD64), but does not bind FcγRIII (CD16). SAPbinds to all three classical Fcγ receptors, with a preference for FcγRIand FcγRII, particularly FCγRI. Although there are conflictingobservations on the binding of CRP to FcγR, both SAP and CRP have beenshown to bind to Fc receptors and initiate intracellular signalingevents consistent with FcγR ligation.

In human blood serum, males normally have approximately 32 μg/ml+/−7μg/ml of SAP, with a range of 12-50 μg/ml being normal. Human femalesgenerally have approximately 24 μg/ml+/−8 μg/ml of SAP in blood serum,with a range of 8-55 μg/ml being normal. In human cerebral spinal fluidthere is normally approximately 12.8 ng/ml SAP in human males andapproximately 8.5 ng/ml in females. Combining male and female data, thenormal SAP level in human serum is 26 μg/ml+/−8 μg/ml with a range of12-55 μg/ml being normal. (The above serum levels are expressed asmean+/−standard deviation.)

IL-12

IL-12 has been previously implicated in fibrosis and fibrosing diseases,but most studies have focused on the role of IL-12 in promoting the Th1immune response or by triggering the production of interferon-γ. Thedirect effects of IL-12 on fibrocyte formation do not appear to havebeen previously recognized.

Laminin-1

Laminins are extracellular matrix proteins involved in movement ofmonocytes from the circulation into tissues. In order for leukocytes toenter tissues, they must cross through endothelial cells and thesurrounding basement membrane of blood vessel wall. This processinvolves the tethering, rolling and stopping of the leukocytes on theendothelial cells. Following adhesion to the endothelial cells,leukocytes then cross between the endothelial cells, through the bloodvessel wall and into the tissues. The process of extravasation of cellsthrough blood vessel walls alters their phenotype and function.

These events are controlled by a series of cell surface adhesionreceptors, including integrins. Integrins bind to a wide variety ofligands, including extracellular matrix proteins (ECM), such asfibronectin, vitronectin, collagen and laminin. Matrix proteins arepresent within the basement of the blood vessel wall, includinglaminins. Laminin are a large family of glycoproteins, with aheterotrimeric structure of α, β and γ chains. The use of different α, βand γ chains leads to the expression of at least 12 different lamininisoforms. Different laminins are expressed at different stages ofdevelopment and at different sites within the body.

Scleroderma

Scleroderma is a non-inherited, noninfectious disease that has a rangeof symptoms. It involves the formation of scar tissue containingfibroblasts in the skin and internal organs. The origin of thefibroblasts is unknown. In mild or early cases of scleroderma, there isa hardening of the skin, fatigue, aches and sensitivity to cold. In moresevere and later stages, there is high blood pressure, skin ulcers,difficulty moving joints, and death from lung scarring or kidneyfailure. Approximately 300,000 people in the U.S. have scleroderma. Thedisease has similarities to lupus and rheumatoid arthritis. There is nocure or significant treatment for scleroderma and even diagnosis isdifficult because there is no clinical test.

Nephrogenic Fibrosing Dermopathy

Nephrogenic fibrosing dermopathy (NFD) is a newly recognizedscleroderma-like fibrosing skin condition. It develops in patients withrenal insufficiency. Yellow scleral plaques and circulatingantiphospholipid antibodies have been proposed as markers of NFD. Dualimmunohistochemical staining for CD34 and pro-collagen in the spindlecells of NFD suggest that the dermal cells of NFD may representcirculating fibrocytes recruited to the dermis. Therefore, inhibition offibrocyte formation may alleviate symptoms of this disease.

Asthma

Asthma affects more than 100 million people worldwide, and itsprevalence is increasing. Asthma appears to be caused by chronic airwayinflammation. One of the most destructive aspects of asthma isremodeling of the airways in response to chronic inflammation. Thisremodeling involves thickening of the lamina reticularis (thesubepithelial reticular basement membrane surrounding airways) due tofibrosis. The airway passages then become constricted due to thethickened airway walls.

The thickened lamina reticularis in asthma patients contains abnormallyhigh levels of extracellular matrix proteins such as collagen I,collagen III, collagen V, fibronectin and tenascin. The source of theseproteins appears to be a specialized type of fibroblast calledmyofibroblasts.

In asthma patients, CD34+/collagen I+ fibrocytes accumulate near thebasement membrane of the bronchial mucosa within 4 hours of allergenexposure. 24 hours after allergen exposure, labeled monocytes/fibrocyteshave been observed to express α-smooth muscle actin, a marker formyofibroblasts. These observations suggest that in asthma patientsallergen exposure causes fibrocytes from the blood to enter thebronchial mucosa, differentiate into myofibroblasts, and then causeairway wall thickening and obstruct the airways. Further, there is acorrelation between having a mutation in the regulatory regions of thegenes encoding monocyte chemoattractant protein 1 or TGFβ-1 and theseverity of asthma. This also suggests that recruitment of monocytes andappearance of myofibroblasts lead to complications of asthma.

Thickening of the lamina reticularis distinguishes asthma from chronicbronchitis or chronic obstructive pulmonary disease and is found evenwhen asthma is controlled with conventional medications. An increasedextent of airway wall thickening is associated with severe asthma. Nomedications or treatments have been found to reduce thickening of thelamina reticularis. However, it appears likely that reducing the numberof myofibroblasts found in the airway walls may reduce thickening orhelp prevent further thickening.

Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a unique type of chronicfibrosing lung disease of unknown etiology. The sequence of thepathogenic mechanisms is unknown, but the disease is characterized byepithelial injury and activation, the formation of distinctivesubepithelial fibroblast/myofibroblast foci, and excessive extracellularmatrix accumulation. These pathological processes usually lead toprogressive and irreversible changes in the lung architecture, resultingin progressive respiratory insufficiency and an almost universallyterminal outcome in a relatively short period of time. While researchhas largely focused on inflammatory mechanisms for initiating thefibrotic response, recent evidence strongly suggests that disruption ofthe alveolar epithelium is an underlying pathogenic event. Given therole played by fibrocytes in wound healing and their known role inairway wall thickening in asthma, it appears likely that overproductionof fibrocytes may be implicated in IPF.

SUMMARY

The present invention may include compositions and methods forsuppressing fibrocytes. In the context of the present invention, theterm “suppressing fibrocytes” refers to one or more of inhibiting theproliferation of fibrocytes, inhibiting the development of fibrocytes,including the development or differentiation of a cell into a fibrocyte,and promoting the development or differentiation of fibrocytes intonon-fibrocytic cell types.

In selected embodiments, fibrocytes may be suppressed in a targetlocation by providing SAP, IL-12, Laminin-1, IgG aggregates,cross-linked IgG, cofactors of any of the above, and any combinationthereof. (Designations for “SAP”, “IL-12”, “Laminin-1” and “IgG” as usedherein also refer to functional fragments of these proteins unless it isclear that such fragments are excluded from the usage in a givencontext.) The target location may be located in vitro or in vivo.

Specifically, the target location may be located in a mammal, such as ahuman patient.

In vivo the target location may include an entire organism or a portionthereof and the composition may be administered systemically or it maybe confined to a particular area, such as an organ or tissue.

The compositions may include those supplied directly or produced intarget location or the same organism as the target location, forinstance through expression of a transgene. These compositions may begiven in amount sufficient to increase concentrations above normallevels or to bring their concentrations up to normal levels or restoretheir normal activity levels. Concentrations or activity of certain ofthese compositions may be increased by stimulating natural production orsuppressing normal degradation.

Suppressing fibrocytes may alleviate symptoms of numerous fibrosingdiseases or other disorders caused by fibrosis. In a specificembodiment, administration of SAP, IL-12, Laminin-1, IgG aggregates,cross-linked IgG, cofactors of any of the above, and/or any combinationthereof may be used to treat the effects of unwanted fibrocytes. Forexample, it may be used to treat fibrosis in the liver, kidney, lung,heart and pericardium, eye, skin, mouth, pancreas, gastrointestinaltract, brain, breast, bone marrow, bone, genitourinary, a tumor, or awound.

Embodiments of the present invention also include assays to detect theability of a sample to suppress fibrocytes. In one embodiment, normalcells (e.g., cells capable of differentiating into fibrocytes, likemonocytes for example) may be supplied with the sample. The sample mayinclude normal SAP. It may also include SAP or a biological fluid from apatient such as a patient with a fibrosing disease, or it may include apotential drug. In another embodiment, the sample may include normal SAPwhile the cells may be derived from a patient and may be abnormal orsuspected of being abnormal. In either type of assay, the effects onfibrocyte suppression may be compared with a normal control to detectany increases or decreases in differentiation as compared to normal.This may indicate the presence or absence of a fibrosing disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed descriptionpresented herein.

FIG. 1 illustrates the effects of serum and plasma on the rapiddifferentiation of fibroblast-like cells.

In FIG. 1A peripheral blood mononuclear cells (PBMC) at 2.5×10⁵ per mlwere cultured in serum-free medium for 3 or 6 days in the presence orabsence of 0.1% human serum and then examined by microscopy for theappearance of fibroblast-like cells. Bar is 100 μm.

In FIG. 1B PBMC at 2.5×10⁵ per ml were cultured in serum-free medium for6 days in dilutions of human plasma. Cells were then air-dried, fixed,stained, and fibrocytes were enumerated by morphology. Results areexpressed as mean±SD of the number of fibrocytes per 2.5×10⁵ PBMCs (n=5experiments). Stars indicate statistically significant differences fromsamples without plasms.

FIG. 2 illustrates the expression of surface molecules onfibroblast-like cells. PBMC were cultured on glass slides in serum-freemedium for 6 days. Cells were air-dried and analyzed byimmunohistochemistry. Monoclonal antibodies used are as indicated, andidentified by biotin-conjugated goat anti-mouse Ig followed byExtrAvidin peroxidase. Cells were counterstained with Mayer'shaematoxylin to identify nuclei. Positive staining was identified bybrown staining, nuclei are counterstained blue. An insert for CD83 wasused to indicate positive staining on a dendritic cell.

FIG. 3 illustrates the characterization of the molecule present inplasma that inhibits fibrocyte differentiation. Citrated plasma wastreated with BaCl₂ and the precipitated material was collected bycentrifugation and dialyzed against 10 mM sodium phosphate containing 10mM EDTA and protease inhibitors. This material was then fractionated byheparin and ion exchange chromatography.

In FIG. 3A fractions were analyzed by PAGE on a 4-20% reducing gel andstained with coomassie blue. M indicates molecular weight markers. Lane1 contained plasma, lane 2 contained BaCl₂ supernatant, lane 3 containedwash 1, lane 4 contained wash 2, lane 5 contained BaCl₂ precipitate,lane 6 contained BaCl₂ precipitate, lane 7 contained heparin flowthrough, lane 8 contained the heparin fraction, lane 9 contained High Qflow through, lane contained the 10 High Q fraction, lane 11 containedthe gel purified fraction. Lanes 1-5 were diluted 1:500 in sodiumphosphate buffer, lanes 6-11 were undiluted.

Active fractions eluted off the High Q ion exchange column and gelslices were analyzed by 4-20% PAGE on a native gel in FIG. 3B and areducing gel in FIG. 3C. NM indicates native gel markers, RM indicatesreduced gel markers. In FIG. 3C lanes 1-3 are control gel samples, lane4 contained active fraction. In FIG. 3D fractions were assessed bywestern blotting, using a rabbit anti-SAP antibody. Lanes 1-11correspond to those in FIG. 3A.

FIG. 4 shows the inhibition of fibrocyte formation by SAP, but not CRPor other plasma proteins. PBMC at 2.5×10⁵ per ml were cultured inserum-free medium for 6 days in the presence of commercially availablepurified SAP (filled square), CRP (open square), Protein S (opendiamond) or C4b (open circle) and then examined for the appearance offibroblast-like cells. Cells were then air-dried, fixed, stained andfibrocytes enumerated by morphology. Results are mean±SD of fibrocytesper 2.5×10⁵ PBMC (n=3 separate experiments).

FIG. 5 shows the effect of depletion of SAP from plasma in a fibrocytedifferentiation assay.

FIG. 5A shows the effect on fibrocyte differentiation of depleting SAPfrom plasma with BioGel agarose beads. Number of fibrocytes found in anassay supplied with either plasma (open square) or BioGel depletedplasma (filled square) at a variety of dilutions is shown. Starsindicate statistically significant differences between the two curves.

FIG. 5B shows the number of fibrocytes formed in an assay performed withno plasma or equal dilutions of plasma, BioGel SAP depleted plasma, oranti-SAP antibody depleted plasma. Stars indicate statisticallysignificant differences.

FIG. 6 shows the effects of various cytokines on monocytedifferentiation into fibrocytes. FIG. 6A shows the effects of a varietyof cytokines. FIG. 6B shows the effects of IL-12 in greater detail.

FIG. 7 shows the effects of extracellular matrix proteins on fibrocyteformation. Extracellular matrix proteins were bound to 96 well tissueculture plates for 18 hours at 4° C. in 50 mM carbonate buffer pH 9.5.ProNectin-F and ProNectin-L were diluted in PBS. Plates were washed inPBS, and incubated for 60 minutes at 37° C. in PBS containing 2% bovineserum albumin, to prevent non-specific binding. Plates were washed withPBS and then tissue culture medium. PBMC were then added and culturedfor 4 days. Results for a variety of extracellular matrix proteins areshown in FIG. 7A. The effects of Laminin-1, Laminin-10/11 andProNectin-L are shown in greater detail in FIG. 7B.

FIG. 8 shows the effects of monomeric IgG on the ability of SAP to bindto monocytes and inhibit their differentiation. PBMC were cultured inserum-free medium in the presence of a range of concentrations ofmonomeric IgG for 60 minutes. SAP, at the concentrations indicated, wasthen added and the cells were cultured for 4 days.

FIG. 9 shows the effects of ligation and cross-linking of Fc receptorson monocyte to fibrocyte differentiation. Soluble immune complexes(ovalbumin-antibody), particulate immune complexes, including opsonisedsheep red blood cells (SRBC) and heat-aggregated IgG were used. In FIG.9A PBMC cultured for 4 days with ovalbumin or anti-ovalbumin mAb alone,or ovalbumin:anti-ovalbumin immune complexes. FIG. 9B shows the effectsof SRBC alone and SRBC opsonised with rabbit anti-SRBC at 20:1 and 40:1SRBC:monocyte ratios. Finally, FIG. 9C shows the effects on PBMC ofheat-aggregated IgG and heat-aggregated F(ab)₂. Stars in 9A and 9Bindicate statistically significant differences.

FIG. 10 shows the effects of anti-FcγR antibodies on monocytedifferentiation. Stars indicate a statistically significant differencefrom control.

FIG. 11 shows the effects of SAP on collagen content in rat lungs.Intra-tracheal injection of bleomycin (Bleo) was used to inducefibrosis. Control rats had saline injected into their tracheas. “+SAP”indicates that rats were given an intravenous injection of 240 μg of ratSAP on days 1, 3, 5, 7 and 9. The animals were euthanized on day 14.Lung tissues were removed and homogenized then assayed for collagencontent. Values are means+/−SEM (n=4). * indicates p<0.05 as determinedby ANOVA.

FIG. 12 shows cross sections of rat lungs after administration of salineor bleomycin with or without SAP. FIG. 12A shows a cryosection of asaline-treated rat lung 14 days after treatment began. FIG. 12B shows acryosection of a bleomycin-treated rat lung 14 days after treatmentbegan. FIG. 12C shows a cryosection of a SAP-treated rat lung alsotreated with bleomycin 14 days after treatment began. The rat wasinjected with 240 μg of purified rat SAP every 2 days for 9 days,starting the day after bleomycin treatment. All three sections werestained with Picrosirius red to label collagen. Bar is 0.5 mm.

FIG. 13 shows the effects of SAP on fibrosis in rat lungs.Intra-tracheal injection of bleomycin (Bleo) was used to inducefibrosis. Control rats had saline injected into their tracheas. “+SAP”indicates that rats were given an intravenous injection of 240 μg of ratSAP on days 1, 3, 5, 7 and 9. The animals were euthanized on day 14.Lung tissues were removed and fibrosis was assessed using a modifiedAshcroft score containing 5 fields per section, and from three separateareas of lung. Zero is a normal lung, 1 is minimal thickening of thealveolar wall, 2 and 3 are increased levels of fibrosis, and 4 is severedistortion of the lung structure with large areas of fibrosis. Valuesare means+/−SEM (n=4). *** indicates p<0.001 as determined by ANOVA.

DETAILED DESCRIPTION Fibrocyte Suppression

The regulation of events leading to fibrosis involves the proliferationand differentiation of fibrocytes. Fibrocytes are a distinct populationof fibroblast-like cells derived from peripheral blood monocytes thatnormally enter sites of tissue injury to promote angiogenesis and woundhealing. Culturing CD14+ peripheral blood monocytes in the absence ofserum or plasma leads to the rapid differentiation of fibrocytes. Thisprocess occurs within 72 hours and is suppressed by the presence ofserum or plasma. The factor in serum that suppresses the rapidappearance of fibrocytes is serum amyloid P (SAP). Further, a cohort ofpatients with the fibrosing disease scleroderma have sera with a poorability to suppress fibrocyte differentiation and exhibit acorrespondingly low level of SAP. These results suggest that low levelsof SAP in the circulation or the peripheral tissues lead to or play apart in pathological processes such as fibrosis. Monocytedifferentiation assays have also revealed that IL-12, Laminin-1 andconjugated IgG molecules also suppress differentiation of monocytes intofibrocytes.

Compositions containing one or more of the fibrocyte formationsuppressors may be used to suppress fibrosis in inappropriate locationsand in fibrosing disorders and chronic inflammatory conditions, interalia.

Compositions may be applied locally or systemically. In specificembodiments, compositions containing SAP may be operable to raise SAPconcentration in target locations to approximately at least 0.5 μg/ml.In humans, I¹²⁵ radiolabelled SAP has been previously administered tostudy patients with amyloidosis. In the treatments, approximately 100 μgof SAP was administered to an adult human. Accordingly, administrationof approximately 100 μg of SAP systemically to an adult human is safe.Higher dosages may also be safe under appropriate conditions.

SAP supplied in certain compositions of the present invention mayinclude the entire SAP protein or a portion thereof, preferably theportion functional in suppression fibrocyte formation. In an exemplaryembodiment, the functional portion of SAP is selected from the regionthat does not share sequence homology with CRP, which has no effect onfibrocyte formation. For instance amino acids 65-89KERVGEYSLYIGRHKVTSKVIEKFP (SEQ ID NO:1) of SAP are not homologous toCRP. Amino acids 170-181 ILSAYQGTPLPA (SEQ ID NO:2) and 192-205IRGYVIIKPLV (SEQ ID NO:3) are also not homologous. Additionally a numberof single amino acid differences between the two proteins are known andmay result in functional differences.

Compositions containing IL-12 may be operable to raise the IL-12concentration in target locations to approximately 0.1 to 10 ng/ml.Compositions containing Laminin-1 may be operable to raise the laminin-1concentration in target locations to approximately 1 to 10 μg/ml.Compositions containing aggregated IgG may be operable to raiseaggregate IgG concentrations in target locations to approximately 100μg/ml. The compositions may also be supplied in combinations or withcofactors. Compositions may be administered in an amount sufficient torestore normal levels, if the composition is normally present in thetarget location, or they may be administered in an amount to raiselevels above normal levels in the target location.

The above compositions may be supplied to a target location from anexogenous source, or they may be made in vivo by cells in the targetlocation or cells in the same organism as the target location. Thesecompositions may be isolated from donated human tissues, includingbiological fluids. They may be also be made as a recombinant protein inbacteria, tissue culture cells, or any other type of cells or tissuesknown to the art, or in whole animals. They may also be madesynthetically or by any other methodology known to the art. If thesecompositions are made in vivo, they may be the expression product of atransgene or they may result from enhancement of production in anexisting in vivo source. Levels of these compositions, if they arenormally present in a target location, may also be raised by reducingtheir normal rates of degradation. Additionally, it may be possible toincrease the fibrocyte suppression ability of these compositions, forinstance by supplying cofactors.

In a specific embodiment, the compositions may include SAP coupled to anagent to prolong its serum half-life or otherwise to facilitate deliveryof the SAP to the area of the fibrosing disease, as opposed to removalby the body as waste. For example, the SAP may be conjugate to abiocompatible polymer such as PEG, a poly(amino acid), or apolysaccharide.

Compositions of the present invention may be in any physiologicallyappropriate formulation. They may be administered to an organismtopically, by injection, by inhalation, orally or by any other effectivemeans.

Disease Targets

The same compositions and methodologies described above to suppressfibrocytes may also be used to treat or prevent conditions resultingfrom inappropriate fibrocyte proliferation or differentiation. Forexample, they may treat or prevent a condition occurring in the liver,kidney, lung, heart and pericardium, eye, skin, mouth, pancreas,gastrointestinal tract, brain, breast, bone marrow, bone, genitourinary,a tumor, or a wound.

Generally, they may treat or prevent fibrosis resulting from conditionsincluding but not limited to rheumatoid arthritis, lupus, pathogenicfibrosis, fibrosing disease, fibrotic lesions such as those formed afterSchistosoma japonicum infection, radiation damage, autoimmune diseases,Lyme disease, chemotherapy induced fibrosis, HIV or infection-inducedfocal sclerosis, failed back syndrome due to spinal surgery scarring,abdominal adhesion post surgery scarring, fibrocystic formations,fibrosis after spinal injury, surgery-induced fibrosis, mucosalfibrosis, peritoneal fibrosis caused by dialysis, andAdalimumab-associated pulmonary fibrosis.

Specifically, in the liver, they may treat or prevent fibrosis resultingfrom conditions including but not limited to alcohol, drug, and/orchemically induced cirrhosis, ischemia-reperfusion injury after hepatictransplant, necrotizing hepatitis, hepatitis B, hepatitis C, primarybiliary cirrhosis, and primary sclerosing cholangitis.

Relating to the kidney, they may treat or prevent fibrosis resultingfrom conditions including but not limited to proliferative andsclerosing glomerulonephritis, nephrogenic fibrosing dermopathy,diabetic nephropathy, renal tubulointerstitial fibrosis, and focalsegmental glomerulosclerosis.

Relating to the lung, they may treat or prevent fibrosis resulting fromconditions including but not limited to pulmonary interstitial fibrosis,sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma,chronic obstructive pulmonary disease, diffuse alveolar damage disease,pulmonary hypertension, neonatal bronchopulmonary dysplasia, chronicasthma, and emphysema. There are several sub-names or synonyms forpulmonary fibrosis including, but not limited to, cryptogenic fibrosingalveolitis, diffuse interstitial fibrosis, idiopathic interstitialpneumonitis, Hamman-Rich syndrome, silicosis, asbestosis, berylliosis,coal worker's pneumoconiosis, black lung disease, coal miner's disease,miner's asthma, anthracosis, and anthracosilicosis.

Relating to the heart and/or pericardium, they may treat or preventfibrosis resulting from conditions including but not limited tomyocardial fibrosis, atherosclerosis, coronary artery restenosis,congestive cardiomyopathy, heart failure, and other post-ischemicconditions.

Relating to the eye, they may treat or prevent fibrosis resulting fromconditions including but not limited to exopthalmos of Grave's disease,proliferative vitreoretinopathy, anterior capsule cataract, cornealfibrosis, corneal scarring due to surgery, trabeculectomy-inducedfibrosis, progressive subretinal fibrosis, multifocal granulomatouschorioretinitis, and other eye fibrosis.

Relating to the skin, they may treat or prevent fibrosis resulting fromconditions including but not limited to Depuytren's contracture,scleroderma, keloid scarring, psoriasis, hypertrophic scarring due toburns, atherosclerosis, restenosis, and psuedoscleroderma caused byspinal cord injury.

Relating to the mouth and/or esophagus, they may treat or preventfibrosis resulting from conditions including but not limited toperiodontal disease scarring, gingival hypertrophy secondary to drugs,and congenital esophageal stenosis.

Relating to the pancreas, they may treat or prevent fibrosis resultingfrom conditions including but not limited to pancreatic fibrosis,stromal remodeling pancreatitis, and stromal fibrosis.

Relating to the gastrointestinal tract, they may treat or preventfibrosis resulting from conditions including but not limited tocollagenous colitis, villous atrophy, crypt hyperplasia, polypformation, fibrosis of Crohn's disease, and healing gastric ulcer.

Relating to the brain, they may treat or prevent fibrosis resulting fromconditions including but not limited to glial scar tissue.

Relating to the breast, they may treat or prevent fibrosis resultingfrom conditions including but not limited to fibrocystic disease anddesmoplastic reaction to breast cancer.

Relating to the bone marrow, they may treat or prevent fibrosisresulting from conditions including but not limited to fibrosis inmyelodysplasia and neoplastic diseases.

Relating to the bone, they may treat or prevent fibrosis resulting fromconditions including but not limited to rheumatoid pannus formation.

Relating to the genitourinary system, they may treat or prevent fibrosisresulting from conditions including but not limited to endometriosis,uterine fibroids, ovarian fibroids, and Peyronie's disease.

Relating to radiation induced damage, they may treat or prevent fibrosisrelated to, but not limited to, treatment of head and neck cancer,ovarian cancer, prostate cancer, lung cancer, gastrointestinal cancer,colon cancer, and breast cancer.

The invention also includes a method of suppressing fibrocytes ortreating or preventing a fibrosing disease or asthma by activating anycomponent of the Fc signaling pathway in cells normally activated bySAP. This pathway is described in detail in Daeron, Marc, “Fc ReceptorBiology”, Annu. Rev. Immunology 15:203-34 (1997). In an exemplaryembodiment, a portion of the pathway that is not shared with othersignaling cascades or only a limited number of non-critical signalingcascades is selected for activation to minimize side-effects.

In a particular embodiment, pulmonary fibrosis or other pulmonaryfibrosing diseases may be treated by administration of SAP. Treatmentmay reduce cellular growth associated with fibrosis and also collagendeposition. Treatment may prevent further fibrosis or reduce the effectsof current fibrosis. SAP may be administered in a dose of approximately1.6 μg/g of bodyweight or in another dose able to approximately doublethe serum concentration of SAP in the patient. Administration may beintravenous and may take place every other day for a selected duration.This dose, method of administration and administration schedule may alsobe useful in treating other fibrosing diseases.

Differentiation Assays

Another aspect of the invention relates to assays to detect the abilityof a sample to suppress fibrocytes. In serum-free medium, normalmonocytes form fibrocytes in two to three days. Normal serum, blood orother biological fluids suppress the formation of fibrocytes from normalmonocytes over a specific dilution range. Thus the assay may be used totest whether a sample can modulate differentiation of monocytes intofibrocytes in serum-free medium. It may also be used to determinewhether sample monocytes differentiate normally into fibrocytes inserum-free medium and if they respond normally to serum, SAP or otherfactors affecting this differentiation.

In a specific embodiment, the assay may be used to determine whether apatient's biological fluid has a decreased or increased ability tosuppress fibrocytes. If suppression by SAP is to be tested, anybiological fluid in which SAP is normally or transiently present may beused in the present invention, including whole blood, serum, plasma,synovial fluid, cerebral spinal fluid and bronchial fluid. A decreasedability of any of these fluids to suppress fibrocytes may be indicativeof a fibrosing disease or the propensity to develop such a disease.

Although in many patients a decreased ability of a biological fluid tosuppress fibrocyte formation may be due to low levels of SAP, this isnot necessarily the case. SAP may be present at normal levels, butexhibit decreased suppressive activity due to defects in the SAP itselfor the absence or presence of a cofactor or other molecule. Methods ofdetermining the more precise nature of the suppression problem, such asuse of ELISAs, electrophoresis, and fractionation will be apparent toone skilled in the art.

The methodology described above may also be used to determine whethercertain potential drugs that affect fibrocyte proliferation ordifferentiation may or may not be appropriate for a patient.

In another specific embodiment, the assay may be used to determine if apatient's cells are able to differentiate into fibrocytes in serum-freemedium and if they respond normally to a biological fluid, SAP oranother composition. More particularly, if a patient with a fibrosingdisease appears to have normal levels of SAP, particularly functionalSAP, it may be advisable to obtain a sample of the patient's cells(e.g., monocytes) to determine if they are able to readily differentiateinto fibrocytes even in the presence of serum or SAP. If the patient'scells are able to differentiate in the presence of normal SAP, then thecells themselves and not any SAP deficiencies may be the cause of thepatient's disease.

This assay may also be used to determine if any drugs are appropriatefor a particular patient.

Finally, in another specific example, the assay may be used to test theeffects of a drug or other composition on a cell's differentiation intofibrocytes. The assay may be used in this manner to identify potentialdrugs designed to modulate fibrocyte formation, or it may be used toscreen for any potential adverse effects of drugs intended for otheruses.

The following examples are included to demonstrate specific embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES Example 1 Inhibition of Fibrocyte Formation

While examining the possible role of cell density in the survival ofperipheral blood T cells, it was observed that in serum-free medium PBMCgave rise to a population of fibroblast-like cells. These cells wereadherent and had a spindle-shaped morphology (FIG. 1A). Approximately0.5-1% of PBMC differentiated into fibroblast-like cells in serum-freemedium, and this occurred in tissue culture treated plasticware andborosilicate and standard glass slides.

The rapid appearance of these cells, within 3 days of culture, wasinhibited by human serum or plasma. To examine this process in moredetail, PBMC were cultured at 5×10⁵ cells per ml in serum-free mediumcontaining increasing concentrations of human plasma for 6 days. Whenplasma was present at concentrations between 10% and 0.5%, thefibroblast-like cells did not differentiate (FIG. 1B). However, at orbelow 0.1% serum, fibroblast-like cells rapidly developed. The activityin the serum that inhibited fibrocyte formation was retained by a 30 kDacutoff spin-filter (data not shown). If serum was heated to 56° C. for30 minutes, the efficacy was reduced 10 fold, and heating to 95° C.abolished the inhibitory activity (data not shown).

These data suggest that that the inhibitory factor is a protein. As theinhibitory factor was present in human serum, it indicated that theactivity was unlikely to be involved with the coagulation system. Theinhibitory factor also appeared to be an evolutionary conserved proteinas bovine, equine, caprine, and rat sera were also able to inhibit theappearance of these fibroblast-like cells (data not shown).

Example 2 Characterization of Fibroblast-Like Cells

The differentiation of these fibroblast-like cells from peripheral bloodsuggested that they might be peripheral blood fibrocytes. Fibrocytes area population derived from peripheral blood monocytes that differentiatein vitro and in vivo into fibroblast-like cells. They rapidly enterwound sites and are capable of presenting antigens to T cells. Theirphenotype is composed of both hematopoietic markers, such as CD45 andMHC class II, and stromal markers, such as collagen I and fibronectin.However in order to identify these cells, PBMC were generally culturedfor 1-2 weeks in medium containing serum.

To characterize whether the cells observed in the system werefibrocytes, PBMC were depleted of T cells with anti-CD3, B cells withanti-CD19, monocytes with anti-CD14 or all antigen presenting cells withanti-HLA class II and then cultured in serum-free conditions for 6 days.Depletion of PBMC with anti-CD3 or anti-CD19 did not depletefibroblast-like cells from PBMC when cultured in serum-free cultures(data not shown). Depletion of antigen presenting cells with anti-HLAclass II or monocytes with anti-CD14 antibody did prevent the appearanceof fibroblast-like cells, indicating that the fibroblast-like cells arederived from monocytes and not a dendritic cell population.

To further characterize the fibroblast-like cells, PBMC were cultured inserum-free medium for 5 days on glass slides. Cells were then air-dried,fixed in acetone and labeled with a variety of antibodies (Table 1 andFIG. 2). Fibrocytes express CD11a, CD11b, CD45, CD80, CD86, MHC classII, collagen I, fibronectin, the chemokine receptors CCR3, CCR5, CCR7,CXCR4 and α-smooth muscle actin. In the above culture conditions, thefibroblast-like cells in the present experiment also expressed all thesemarkers. Fibrocytes are negative for CD1a, CD3, CD19, CD38 and vWF, aswere the fibroblast-like cells in the present experiment. Based on thesedata it appears that the fibroblast-like cells observed in theexperiments were fibrocytes. Further experiments were conducted toextend this phenotype. In the above conditions, the fibrocytes expressedseveral β1 integrins including α1 (CD49a), α2 (CD49b), α5 (CD49e), β1(CD29) and β3 (CD61) along with high levels of β2 (CD18), but werenegative for α3, α4, α6 α4β7, αE and CLA (FIG. 2 and Table 1).

TABLE 1 Expression of surface markers on Fibrocytes Marker AlternativeName Fibrocyte Expression CD11a LFA-1 positive CD11b Mac-1 positiveCD11c positive CD13 positive CD18 β2 integrin positive CD29 β1 integrinpositive CD34 positive CD40 weak positive CD45 LCA positive CD49a α1integrin weak positive CD49b α2 integrin negative CD49e α5 integrinpositive CD51 positive CD54 ICAM-1 positive CD58 LFA-3 positive CD61 β3integrin positive CD80 B7-1 weak positive CD86 B7-2 positive CD105Endoglin positive CD148 positive MHC class II positive CD162 PSGL-1positive CCR1 weak positive CCR3 weak positive CCR4 weak positive CCR5weak positive CCR7 weak positive CCR9 weak positive CXCR1 positive CXCR3positive CXCR4 weak positive Collagen I positive Collagen III positiveFibronectin positive α Smooth Muscle Actin positive Vimentin positiveCD1a negative CD3 negative CD10 negative CD14 negative CD19 negativeCD25 negative CD27 negative CD28 negative CD38 negative CD49c α3integrin negative CD49d α4 integrin negative CD49f α6 integrin negativeCD69 negative CD70 CD27-L negative CD90 negative CD103 αE integrinnegative CD109 negative CD154 CD40-L negative α4β7 negative CLA negativeCCR2 negative CCR6 negative CXCR2 negative CXCR5 negative CXCR6 negativeCytokeratin negative vWF negative

To obtain the data in Table 1, PBMC were cultured in the wells of 8 wellglass slides at 2.5×10⁵ cells per ml (400 μl per well) in serum-freemedium for 6 days. Cells were then air dried, fixed in acetone andstained by immunoperoxidase. Cells were scored positive or negative forthe indicated antigens, compared to isotype-matched control antibodies.

Example 3 Characterization of the Fibrocyte Inhibitory Factor

The initial characterization of the serum factor that prevents rapidfibrocyte differentiation indicated that the factor was aheparin-binding molecule that eluted off an ion exchange column (High Q)as one of four proteins. By sequencing tryptic fragments of protein in aband cut from a native gel, one of these proteins was identified asC4b-binding protein (C4BP). C4b-binding protein is a 570 kDa protein,composed of seven alpha chains (70 kDa) and usually a single beta chain(40 kDa), which is involved in regulating the decay of C4b and C2acomponents of the complement system. C4BP also interacts with thevitamin K-dependent anticoagulant protein S. The C4BP/Protein S complexcan be purified from serum or plasma using BaCl₂ precipitation.

To assess whether C4BP, or an associated protein, was the factorresponsible for inhibiting fibrocyte differentiation, citrated plasmawas treated with BaCl₂. The inhibitory factor was present in the BaCl₂precipitate (FIG. 3 and Table 2). This fraction was applied to a heparincolumn and the fractions, eluted by increasing concentrations of NaCl,were assessed for their ability to inhibit monocyte to fibrocytedifferentiation in serum free medium. The active factor was eluted offthe heparin column in a peak at 200 mM NaCl (FIG. 3 and Table 2). Aslight increase in the yield suggested that this step may have removed afactor that slightly interfered with the activity of the factor.

The fractions from the 200 mM peak were pooled and further fractionatedby High Q ion exchange chromatography. A small peak eluting at 300 mMNaCl contained activity that inhibited fibrocyte differentiation.Analysis of the proteins present in this fraction indicated that themajor band was a 27 kDa protein. Although the ion exchangechromatography led to a reduction in the amount of SAP recovered (FIG.3A, lanes 8-10 and FIG. 3D, lane 8-10) this step did remove severalcontaminating proteins. After the ion exchange step the only discernablecontaminant was albumin at 65 kDa (FIG. 3A, lane 10).

The high Q fraction was concentrated and fractionated by electrophoresison a non-denaturing polyacrylamide gel, followed by elution of thematerial in gel slices. A single band that migrated at approximately 140kDa was able to inhibit differentiation (FIG. 3B). This band had amolecular weight of 27 kDa on a reducing polyacrylamide gel, suggestingthat the native conformation of the protein was a pentamer (FIG. 3C).This band was excised from the gel, digested with trypsin and analyzedby MALDI mass spectrometry. Three major and two minor peptides wereidentified: VFVFPR (SEQ ID NO:4), VGEYSLYIGR (SEQ ID NO:5), AYSLFSYNTQGR(SEQ ID NO:6), QGYFVEAQPK (SEQ ID NO:7) and IVLGQEQDSYGGK (SEQ ID NO:8).These sequences exactly matched amino acid sequences 8-13, 68-77, 46-57,121-130 and 131-143 of serum amyloid P.

To confirm that the active fractions contained SAP, fractions collectedfrom column chromatography were analyzed by western blotting (FIG. 3D).The presence of SAP at 27 kDa was detected in all fractions thatinhibited fibrocyte differentiation (FIG. 3D, lanes 6, 8, 10 and 11). Aconsiderable amount of SAP was present in the supernatant from the BaCl₂precipitation step indicating that this procedure was inefficient, withthe recovery of only approximately 10-15% of the fibrocyte inhibitoryactivity in the BaCl₂ pellet (FIG. 3A, lane 2). In order to remove theknown problem of anti-SAP antibodies binding to immunoglobulins whenused with western blotting, the antibody was pre-incubated with humanIgG bound to agarose. Fractions were also analyzed for the presence ofCRP, C4BP and protein S. Western blotting indicated that C4BP andProtein S were present in plasma, and in the barium precipitation, butwere absent from the active fractions collected from heparinchromatography (data not shown).

TABLE 2 Recovery of protein and fibrocyte inhibitory activity fromfractionated human plasma Total Volume Protein protein Yield (ml)(mg/ml) (mg) (%) Plasma 250 70 17,500 100 BaCl₂ 240 60 14,400 82.3supernatant BaCl₂ 31 1 31 0.18 precipitate Heparin 4.3 0.25 1.075 0.006fraction High Q 1.96 0.05 0.098 0.00056 fraction Gel slice 0.075 0.0250.0018 0.00001 Total Specific Activity activity Yield activity (U/ml)(U) (%) (U/mg) Plasma 10,000 2.5 × 10⁶ 100 143 BaCl₂ 6,666 1.6 × 10⁶ 64111 supernatant BaCl₂ 1,666 5.1 × 10⁴ 2 1,645 precipitate Heparin 5002,150 0.086 2000 fraction High Q 400 720 0.029 7,300 fraction Gel slice2000 150 0.006 80,000

Plasma was fractionated by BaCl₂ precipitation, heparin and ion exchangechromatography. Protein concentrations were assessed byspectrophotometry at 280 nm. Inhibition of fibrocyte differentiation wasassessed by morphology. The fibrocyte inhibitory activity of a samplewas defined as the reciprocal of the dilution at which it inhibitedfibrocyte differentiation by 50%, when added to serum-free medium.

SAP may also be detected by ELISA using the following methodology:

Maxisorb 96 well plates (Nalge Nunc International, Rochester, N.Y.) werecoated overnight at 4° C. with monoclonal anti-SAP antibody (SAP-5,Sigma) in 50 mM sodium carbonate buffer pH 9.5. Plates were thenincubated in Tris buffered saline pH 7.4 (TBS) containing 4% BSA (TBS-4%BSA) to inhibit non-specific binding. Serum and purified proteins werediluted to 1/1000 in TBS-4% BSA, to prevent SAP from aggregating andincubated for 60 minutes at 37° C. Plates were then washed in TBScontaining 0.05% Tween-20. Polyclonal rabbit anti-SAP antibody(BioGenesis) diluted 1/5000 in TBS-4% BSA was used as the detectingantibody. After washing, 100 pg/ml biotinylated goat F(ab)₂ anti-rabbit(Southern Biotechnology Inc.) diluted in TBS-4% BSA was added for 60minutes. Biotinylated antibodies were detected by ExtrAvidin peroxidase(Sigma). Undiluted peroxidase substrate 3,3,5,5-tetramethylbenzidine(TMB, Sigma) was incubated for 5 minutes at room temperature before thereaction was stopped by 1N HCl and read at 450 nm (BioTek Instruments,Winooska, Vt.). The assay was sensitive to 200 pg/ml.

Example 4 Specificity of Serum Amyloid P

Serum amyloid P is a constitutive plasma protein and is closely relatedto CRP, the major acute phase protein in humans. To assess whether otherplasma proteins could also inhibit the differentiation of fibrocytes,PBMC were cultured in serum-free medium in the presence of commerciallyavailable purified SAP, CRP, C4b or Protein S. The commerciallyavailable SAP was purified using calcium-dependent affinitychromatography on unsubstituted agarose. Of the proteins tested, onlySAP was able to inhibit fibrocyte differentiation, with maximalinhibitory activity at 10 μg/ml (FIG. 4). A dilution curve indicatedthat the commercially available SAP has approximately 6.6×10³ units/mgof activity (FIG. 4). Serum and plasma contain between 30-50 μg/ml SAP.Fibrocytes began to appear at a plasma dilution of 0.5%, which would beapproximately 0.15-0.25 μg/ml SAP, which is comparable to the thresholdconcentration of purified SAP. The data showing that SAP purified usingtwo different procedures inhibits fibrocyte differentiation stronglysuggests that SAP inhibits fibrocyte differentiation.

Although these data indicate that SAP is capable of inhibiting fibrocytedevelopment and SAP purifies in a manner that indicates that it is theactive factor in plasma, it was not determined whether depletion of SAPfrom plasma and serum would negate the inhibition. Accordingly, SAP wasdepleted from plasma using agarose beads (BioGel A, BioRad). Plasma wasdiluted to 20% in 100 mM Tris pH 8, 150 mM NaCl, 5 mM CaCl₂ buffer andmixed with 1 ml agarose beads for 2 hours at 4° C. Beads were thenremoved by centrifugation and the process repeated. This depleted plasmawas then assessed for its ability to inhibit fibrocyte differentiation.The control plasma diluted to 20% in 100 mM Tris pH 8, 150 mM NaCl, 5 mMCaCl₂ buffer had a similar dilution curve to that observed withuntreated plasma. In contrast, the bead-treated plasma was less able toinhibit fibrocyte differentiation at intermediate levels of plasma.These data, along with the ability of purified SAP to inhibit fibrocytedifferentiation, strongly suggest that SAP is the active factor in serumand plasma that inhibits fibrocyte differentiation. (See FIG. 5).

Plasma was also depleted of SAP using protein G beads coated withanti-SAP antibodies. Removal of SAP led to a significant reduction inthe ability of plasma to inhibit fibrocyte differentiation compared withplasma, or plasma treated with beads coated with control antibodies(p<0.05) (FIG. 5B). The beads coated with control antibodies did removesome of the fibrocyte-inhibitory activity from plasma, but this was notsignificantly different from cells cultured with plasma. This probablyreflects SAP binding to the agarose in the protein G beads. These data,together with the ability of purified SAP to inhibit fibrocytedifferentiation, strongly suggest that SAP is the active factor in serumand plasma that inhibits fibrocyte differentiation.

Example 5 Antibodies and Proteins

Purified human CRP, serum amyloid P, protein S and C4b were purchasedfrom Calbiochem (San Diego, Calif.). Monoclonal antibodies to CD1a, CD3,CD11a, CD11b, CD11c, CD14, CD16, CD19, CD34, CD40, Pan CD45, CD64, CD83,CD90, HLA-DR/DP/DQ, mouse IgM, mouse IgG1 and mouse IgG2a were from BDPharmingen (BD Biosciences, San Diego, Calif.). Chemokine receptorantibodies were purchased from R and D Systems (Minneapolis, Minn.).Rabbit anti-collagen I was from Chemicon International (Temecula,Calif.), monoclonal C4b-binding protein was from Green MountainAntibodies (Burlington, VE), sheep anti human C4b-binding protein wasfrom The Binding Site (Birmingham, UK), monoclonal anti-CRP was fromSigma (St. Louis, Mo.). Polyclonal rabbit anti-protein S was fromBiogenesis (Poole, Dorset, UK).

Example 6 Cell Separation

Peripheral blood mononuclear cells were isolated from buffy coats (GulfCoast Regional Blood Center, Houston, Tex.) by Ficoll-Paque (AmershamBiosciences, Piscataway, N.J., USA) centrifugation for 40 minutes at400×g. Depletion of specified leukocyte subsets was performed usingnegative selection using magnetic Dynabeads (Dynal Biotech Inc., LakeSuccess, N.Y.). Briefly, PBMC were incubated with primary antibodies for30 minutes at 4° C. Cells were then washed and incubated with Dynabeadscoated with goat anti-mouse IgG for 30 minutes, before removal ofantibody-coated cells by magnetic selection. This process was repeatedtwice. The negatively selected cells were routinely in excess of 98%pure as determined by monoclonal antibody labeling.

Example 7 Cell Culture and Fibrocyte Differentiation Assay

Cells were incubated in serum-free medium: RPMI (GibcoBRL Life,Invitrogen, Carlsbad, Calif., USA) supplemented with 10 mM HEPES(GibcoBRL/Life), 2 mM glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin, 0.2% bovine serum albumin (BSA, Sigma), 5 μg/ml insulin(Sigma), 5 μg/ml iron-saturated transferrin (Sigma) and 5 ng/ml sodiumselenite (Sigma). Normal human serum (Sigma), normal human plasma (GulfCoast Regional Blood Center) or fetal calf serum (Sigma), columnfractions, sera and synovial fluid from patients or purified proteinswere added at the stated concentrations. Patient samples were obtainedfrom a repository available to researchers at University of TexasMedical School at Houston. This repository keeps patient informationconfidential, and meets all NIH guidelines.

PBMC were cultured in 24 or 96 well tissue culture plates in 2 ml or 200μl volumes respectively (Becton Dickinson, Franklin Lakes, N.J.) at2.5×10⁵ cells per ml in a humidified incubator containing 5% CO₂ at 37°C. for the indicated times. Fibrocytes in 5 different 900 μm diameterfields of view were enumerated by morphology in viable cultures asadherent cells with an elongated spindle-shaped morphology as distinctfrom small lymphocytes or adherent monocytes. Alternatively cells wereair dried, fixed in methanol and stained with hematoxylin and methyleneblue (Hema 3 Stain, VWR, Houston, Tex.). Fibrocytes were counted usingthe above criterion and the presence of an oval nucleus. Enumeration offibrocytes was performed on cells cultured for 6 days in flat-bottomed96 well plates, with 2.5×10⁴ cells per well. In addition, fibrocyteidentity was confirmed by immunoperoxidase staining (see below). Thefibrocyte inhibitory activity of a sample was defined as the reciprocalof the dilution at which it inhibited fibrocyte differentiation by 50%,when added to serum-free medium.

Example 8 Purification and Characterization of Serum and Plasma Proteins

100 ml of frozen human serum or plasma was thawed rapidly at 37° C. and1×“Complete” protease inhibitor (Roche, Indianapolis, Ind., USA), 1 mMbenzamidine HCl (Sigma) and 1 mM Pefabloc (AEBSF:4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride, Roche) wereadded. All subsequent steps were performed on ice or at 4° C. Bariumcitrate adsorption of plasma was performed as described previously. Theprecipitate was collected by centrifugation at 10,000×g for 15 minutes,resuspended in 20 ml of 100 mM BaCl₂ plus inhibitors and recentrifuged.After two rounds of washing, the pellet was resuspended to 20 ml in 10mM sodium phosphate buffer pH 7.4 containing 5 mM EDTA and 1 mMbenzamidine HCl and dialyzed for 24 hours against three changes of 4liters of the same buffer.

Chromatography was performed using an Econo system (Bio-Rad, Hercules,Calif.) collecting 1 ml samples with a flow rate of 1 ml/min. Thedialyzed barium citrate precipitate was loaded onto a 5 ml Hi-TrapHeparin column (Amersham Biosciences) and the column was washedextensively in 10 mM sodium phosphate buffer pH 7.4 until the absorbanceat 280 nm returned to baseline. Bound material was eluted with a steppedgradient of 15 mls each of 100, 200, 300 and 500 mM NaCl in 10 mM sodiumphosphate buffer pH 7.4. The fractions that inhibited monocyte tofibrocyte differentiation eluted at 200 mM NaCl. These were pooled (2ml) and loaded onto a 5 ml Econo-Pak High Q column. After washing thecolumn in 10 mM phosphate buffer, the bound material was eluted with thestepped gradient as above, with the active fraction eluting at 300 mMNaCl.

Active fractions from the High Q chromatography were concentrated to 200μl using Aquacide II (Calbiochem) and then loaded onto a 4-20% nativepolyacrylamide gels (BMA, BioWhittaker, Rockland, Me.) as describedpreviously. After electrophoresis, gel lanes were cut into 5 mm slices,mixed with 200 μl 20 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA pH 7.4containing 1 mM benzamidine HCl, crushed with a small pestle in aneppendorf tube and placed on an end-over-end mixer at 4° C. for 3 days.Proteins that eluted from the gel were analyzed for activity. To obtainamino acid sequences, proteins eluted from the gel slices were loadedonto a 4-20% gel with 100 μM thioglycolic acid in the upper chamber(Sigma). After electrophoresis the gel was rapidly stained with Coomasiebrilliant blue, destained, and the bands excised off the gel. Amino acidsequencing was performed by Dr Richard Cook, Protein SequencingFacility, Department of Immunology, Baylor College of Medicine.

Example 9 Western Blotting

For western blotting, plasma and serum samples were diluted 1:500 in 10mM sodium phosphate pH 7.4. Fractions from heparin and High Q columnswere not diluted. Samples were mixed with Laemmeli's sample buffercontaining 20 mM DTT and heated to 100° C. for 5 minutes. Samples wereloaded onto 4-20% Tris/glycine polyacrylamide gels (Cambrex). Samplesfor native gels were analyzed in the absence of DTT or SDS. Proteinswere transferred to PVDF (Immobilon P, Millipore, Bedford, Mass.)membranes in Tris/glycine/SDS buffer containing 20% methanol. Filterswere blocked with Tris buffered saline (TBS) pH 7.4 containing 5% BSA,5% non-fat milk protein and 0.1% Tween 20 at 4° C. for 18 hours. Primaryand biotinylated secondary antibodies were diluted in TBS pH 7.4containing 5% BSA, 5% non-fat milk protein and 0.1% Tween 20 usingpre-determined optimal dilutions (data not shown) for 60 minutes.ExtrAvidin-peroxidase (Sigma) diluted in TBS pH 7.4 containing 5% BSAand 0.1% Tween 20 was used to identify biotinylated antibody andchemiluminescence (ECL, Amersham Biosciences) was used to visualize theresult.

Example 10 Immunohistochemistry

Cells cultured on 8 well glass microscope slides (Lab-Tek, Nalge NuncInternational, Naperville, Ill.) were air dried before fixation inacetone for 15 minutes. Endogenous peroxidase was quenched for 15minutes with 0.03% H₂O₂ and then non-specific binding was blocked byincubation in 2% BSA in PBS for 60 minutes. Slides were incubated withprimary antibodies in PBS containing 2% BSA for 60 minutes.Isotype-matched irrelevant antibodies were used as controls. Slides werethen washed in three changes of PBS over 15 minutes and incubated for 60minutes with biotinylated goat anti-mouse Ig (BD Pharmingen). Afterwashing, the biotinylated antibodies were detected by ExtrAvidinperoxidase (Sigma). Staining was developed with DAB (Diaminobenzadine,Sigma) for 3 minutes and counterstained for 30 seconds with Mayer'shaemalum (Sigma).

Example 11 Expression of Surface Makers on Fibrocytes

PBMC were cultured in the wells of 8 well glass slides at 2.5×10⁵ cellsper ml (400 μl per well) in serum-free medium for 6 days. Cells werethen air dried, fixed in acetone and stained by immunoperoxidase. Cellswere scored positive or negative for the indicated antigens, compared toisotype-matched control antibodies.

Example 12 Recovery of Protein and Fibrocyte Inhibitory Activity fromFractionated Human Plasma

Plasma was fractionated by BaCl₂ precipitation, heparin and ion exchangechromatography. Protein concentrations were assessed byspectrophotometry at 280 nm. Inhibition of fibrocyte differentiation wasassessed by morphology. The fibrocyte inhibitory activity of a samplewas defined as the reciprocal of the dilution at which it inhibitedfibrocyte differentiation by 50%, when added to serum-free medium.

Example 13 IL-12

Experiments have shown that IL-12 is capable of promoting fibrocytedifferentiation in vitro.

Specifically, peripheral blood mononuclear cells were cultured inserum-free medium in the presence of various cytokines (See FIG. 6A).Concentrations of IL-12 above approximately 5 ng/ml inhibited the numberof fibrocytes in culture. (See FIG. 6B.) This indicates that IL-12 iscapable of suppressing the differentiation of fibrocyte precursors intomature fibrocytes.

Example 14 Laminin-1

The process of crossing the endothelium and basement membrane inducesactivation and differentiation signals for monocytes. Therefore,experiments were performed to determine if extracellular matrix proteinshad an effect on the differentiation of fibrocytes. Extracellular matrixproteins were bound to 96 well tissue culture plates for 18 hours at 4°C. in 50 mM carbonate buffer pH 9.5. ProNectin-F and ProNectin-L werediluted in PBS. Plates were washed in PBS, and incubated for 60 minutesat 37° C. in PBS containing 2% bovine serum albumin, to preventnon-specific binding. Plates were washed with PBS and then tissueculture medium. PBMC were then added and cultured for 4 days.Differentiation of fibrocytes was unaffected by culturing on a widevariety of ECM proteins, including collagens, fibronectin andvitronectin. However, culturing PBMC with either laminin-1(Sigma-Aldrich, St. Louis, Mo.) or ProNectin-F (Sanyo ChemicalIndustries Inc, Kyoto, Japan) led to a significant reduction in thenumber of fibrocytes (See FIG. 7A) (p<0.0001). ProNectin-F is aconstruct of silk protein and repeats of the canonical RGD adhesionsequence from fibronectin. ProNectin-L is a similar construct toProNectin-F, with the amino acid sequence IKVAV, from the α1 chain oflaminin.

Additional experiments were performed to determine whether other lamininproteins could suppress fibrocyte differentiation. Laminin 10/11(Chemicon, Temecula, Calif.) a second commercially available laminin,was not capable of inhibiting fibrocyte differentiation, compared tolaminin-1. (See FIG. 7B)

This data suggests that sequences specific to laminin-1, outside theIKVAV region, and absent from laminin-10 and -11, may be responsible forthe suppressive effect on fibrocyte differentiation.

Example 15 Antibody Studies

SAP and CRP augment phagocytosis and bind to Fγ receptors on a varietyof cells. CRP binds with a high affinity to FcγRII (CD32), a loweraffinity to FcγRI (CD64), but does not bind FcγRIII (CD16). SAP binds toall three classical Fcγ receptors, with a preference for FcγRI andFcγRII. Monocytes constitutively express FcγRI. Because this receptorbinds monomeric IgG, it is saturated in vivo. In order to determinewhether the presence of monomeric human IgG could prevent SAP frominhibiting fibrocyte differentiation, PBMC were cultured in serum-freemedium in the presence of a range of concentrations of monomeric IgG for60 minutes. SAP, at the concentrations indicated in FIG. 8A, was thenadded and the cells were cultured for 4 days. As described in the aboveexamples, 2.5 μg/ml SAP in the absence of IgG strongly inhibitedfibrocyte differentiation. (See FIG. 8A.) Monomeric IgG in a range from0.1 to 1000 μg/ml, which corresponds to approximately 0.001 to 10% serumrespectively, had little effect on the suppression of fibrocyteformation by SAP.

To determine whether ligation and cross-linking of Fc receptors couldalso influence monocyte to fibrocyte differentiation, three test sampleswere used; soluble immune complexes (ovalbumin-antibody), particulateimmune complexes, including opsonised SRBC and heat-aggregated IgG. PBMCcultured for 4 days with ovalbumin or anti-ovalbumin mAb showed that thetwo proteins alone had a modest effect on the differentiation ofmonocytes compared to cultures where no reagent was added. (See FIG.9A.) However, the addition of ovalbumin:anti-ovalbumin immune complexesled to a significant reduction in the number of differentiatedfibrocytes (See FIG. 9A). A similar effect was observed when PBMC werecultured with opsonised SRBC. SRBC opsonised with rabbit anti-SRBC at20:1 and 40:1 SRBC:monocyte ratios significantly suppressed fibrocytedifferentiation as compared to cells cultured with SRBC alone (See FIG.9B). Finally, PBMC cultured with heat-aggregated IgG, but notheat-aggregated F(ab)₂, also showed potent inhibition of fibrocytedifferentiation (See FIG. 9C.) Together these data suggest that ligationand cross-linking of Fc receptors is suppressor of monocyte to fibrocytedifferentiation.

The observation that immune complexes inhibit fibrocyte differentiationsuggests that one or more FcγR influences fibrocyte differentiation. Toexamine the role of FcγR in fibrocyte differentiation PBMC were culturedin the presence or absence of blocking antibodies to FcγRI (CD64),FcγRII (CD32) or FcγRIII (CD16) before the addition of SAP, or as acontrol CRP. When samples were pre-incubated with a blocking mAb foreach of the three FcγR, SAP was later able to modestly suppressfibrocyte differentiation. However, in the absence of exogenously addedSAP, the FcγRI (CD64) blocking mAb had a profound effect on fibrocytedifferentiation. Incubation of PBMC with blocking mAb to FcγRI, but notFcγRII or FcγRIII, promoted fibrocyte differentiation as compared tocells cultured with isotype-matched control mAb or cells cultured withno mAb (P<0.01) (See FIG. 10). These data suggested that SAP or IgG,might have been produced by some cells in the culture system over 4days, or that SAP or IgG was retained by cells from the blood. Westernblotting failed to show the presence of SAP or IgG after cells had beencultured for 4 days in vitro. This suggests that the FcγRI blocking mAbhas a direct effect on fibrocyte differentiation or that SAP or IgG wereonly present during the early time points of the cell culture.

Example 16 Pulmonary Fibrosis

To determine the effects of SAP in treatment of a fibrosing disease,pulmonary fibrosis was selected as a model. Pulmonary fibrosis wasinduced in rats (Sprague Dawley, containing surgically implanted jugularcathethers, Charles River Laboratories, Wilmington, Mass.) by injectionof bleomycin into their lungs. Bleomycin is an antineoplastic agentthat, when injected into the airway, causes fibrosis in the lungs of ananimal. It is a standard way to study lung fibrosis. (Crouch, E. 1990.Pathobiology of pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol259:L159-L184.)

To induce fibrosis, rats were anesthetized by 4% isoflurane, maintainedwith 2.5% isoflurane by non-rebreather mask, and monitored to ensure anappropriate surgical plane of anesthesia was achieved and maintained.The ventral side of the neck was shaved and disinfected with anethanol/iodine solution. A vertical midline incision was made in theventral side of the neck, the neck muscles were retracted, and thetrachea was exposed. 300 microliters of a 3.3 U/ml solution (1 unit) ofbleomycin (Calbiochem/EMD Biosciences, San Diego, Calif.) in sterile0.9% saline was injected with a syringe and a 26-gauge needle into thelumen of the trachea. Control rats had saline injected. The incision wasclosed with two or three sutures. This procedure follows that publishedby Underwood et al. (2000. SB 239063, a p38 MAPK inhibitor, reducesneutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung. Am JPhysiol Lung Cell Mol Physiol 279:L895-L902.) During the procedure andpostoperatively the animal was maintained under a heating lamp, and thenplaced back in its cage once it had fully recovered. During theprocedure the animal was checked to ensure it was: i) was breathingregularly, ii) had pink ears and mucous membranes, iii) did not withdrawits foot when its toes were pinched, and iv) did not blink when the eyeor eyelid was touched.

Native rat SAP was isolated from commercially available rat serum(Gemini BioProducts, Woodland, Calif.). To purify the rat SAP, publishedpurification techniques using calcium-dependent binding tophosphoethanolamine-conjugated agarose were followed. (de Beer, F. C.,M. L. Baltz, E. A. Munn, A. Feinstein, J. Taylor, C. Bruton, J. R.Clamp, and M. B. Pepys. 1982. Isolation and characterization ofC-reactive protein and serum amyloid P component in the rat. Immunology45:55-70; Pepys, M. B., D. R. Booth, W. L. Hutchinson, J. R. Gallimore,P. M. Collins, and E. Hohenester. 1997. Amyloid P component. A criticalreview. Amyloid. 4:274-295.) Both native and SDS-polyacrylamide gelelectrophoresis were used to assay the purity of the preparation. Beforeeach experiment, the monocyte to fibrocyte differentiation inhibitingactivity of the SAP preparation was assayed using rat monocytes. Toavoid contamination of the rat SAP that was used for injection,pyrogen-free solutions and sterile plasticware and tubing were used forthe preparation. Endotoxin levels were tested using the a Limulusamebocyte lysate assay kit (E-Toxate, Sigma-Aldrich, St. Louis, Mo.There were no contaminated preparations.

Some of the rats were injected with purified rat SAP intravenously via ajugular catheter implanted by the vendor. The protein was formulated inphysiological saline (0.9% NaCl) and passed through a 0.2 micron filterbefore administration. The dose was 240 micrograms in 0.1 milliliter andwas administered five times over the course of 9 days. This SAPinjection schedule does not affect weight gain, respiration, pulseoximetry, spleen mass, or the appearance of organs at autopsy.

Every two days all the rats were weighed and ˜100 μl of blood wascollected from the jugular cannula. Serum was used to verify that theinjections had increased serum SAP levels by monitoring levels of ratSAP. Serum SAP levels were assayed by western blots (Polyclonal anti-ratSAP, R and D Systems). The first group of four rats was the control, andthe second group of four rats were injected with 240 μg of purified ratSAP via the jugular cannula every two days beginning on day 1 afterweighing and taking a blood sample. The third group of four had lungfibroses induced by bleomycin treatment on day 0, and were injected withsaline via the jugular cannula every two days after removing the bloodsample. The fourth group was injected via the jugular cannula, likegroup 2, with 240 μg of purified rat SAP every two days beginning on day1 and had lung fibrosis induced with bleomycin on day 0. The ratsweighed approximately 150 g each. Thus, approximately 1.6 μg/g wasadministered in each dose. A 150 g rat normally has approximately 8 mlof serum with a SAP concentration of approximately 30 μg/ml.Accordingly, a 240 μg dose approximately doubled the serum concentrationof SAP. The animals were sacrificed on day 14. The injection schedulefor each group of rats is provided in Table 3.

TABLE 3 Injection Schedule for Four Groups of Rats Day Day Day Day Group0 1 3 Day 5 Day 7 Day 9 14 1 Saline Inject Inject Inject Inject Injectsacrifice into saline saline saline saline saline lungs 2 Saline InjectInject Inject Inject Inject sacrifice into SAP SAP SAP SAP SAP lungs 3Bleomycin Inject Inject Inject Inject Inject sacrifice into salinesaline saline saline saline lungs 4 Bleomycin Inject Inject InjectInject Inject sacrifice into SAP SAP SAP SAP SAP lungs

Following euthanasia, lungs were perfused with phosphate-buffered salineto remove blood. One lung was weighed and homogenized. An aliquot of thehomogenate was used to measure collagen using the Sircol collagen assay(Newtonabbey, NI, UK). These collagen measurements are summarized inFIG. 11. Specifically, collagen content in the lungs of ratsadministered bleomycin alone was quite high compared to that of ratsadministered only saline (normal). In contrast, rats administeredbleomycin and SAP showed far less collagen than rats that receivedbleomycin, indicating that SAP helps prevent the development of fibrosisin the lungs and the accompanying accumulation of collagen.

Also of interest, SAP alone may also decrease collagen as compared tonormal. This indicates that SAP may also have the potential to treatexisting fibrosis by reducing collagen.

Tissue from the other lung was embedded in OCT (Sakura Finetek,Torrance, Calif.) and frozen. Cryosections were mounted on SuperfrostPlus (VWR, West Chester, Pa.) slides. Cryosections were stained forcollagen with Picrosirius red (Polysciences Inc., Warrington, Pa.), at 1mg/ml in saturated picric acid. (Junqueira, L. C., G. Bignolas, and R.R. Brentani. 1979. Picrosirius staining plus polarization microscopy, aspecific method for collagen detection in tissue sections. Histochem. J11:447-455.) Fibrosis was assessed using a modified Ashcroft scale,where 0 is normal lung and 4 is severe distortion of the lung structure,with large fibrotic areas. (Ashcroft, T., J. M. Simpson, and V.Timbrell. 1988. Simple method of estimating severity of pulmonaryfibrosis on a numerical scale. J Clin Pathol 41:467-470.) Using a 4×objective, 10 random fields were counted from lung sections taken fromthe top, middle and lower portions of each lung.

Sample lung sections are provided in FIG. 12. FIG. 12A shows the crosssection of lung from a rat in group 1. This lung section has a lacypattern of cells characteristic of a normal lung. FIG. 12B shows thecross section of lung from a rat in group 3, which received bleomycin,but did not receive any SAP. This section shows that the lung has filledwith cells and contains deposits of collagen which stain dark. Thispattern is typical of rats and mice treated with bleomycin and alsofibrotic human lungs. In contrast, FIG. 12C shows the cross section of alung from a rat in group 4. This rat received bleomycin, but alsoreceived SAP. As a result of the SAP, this lung retained a very normallacy appearance. There is no filling with cells and only a few, smallcollagen foci of collagen deposition. Thus, the administration of SAPappears to have prevented the development of pulmonary fibrosis in rats.

FIG. 13 summarizes the lung section data for all four groups of rats.Rats receiving bleomycin had a very high fibrosis score using a modifiedAshcroft score as compared to rats receiving saline only (normal). Thisfibrosis score was halved by the co-administration of SAP withbleomycin, demonstrating the ability of SAP to inhibit pulmonaryfibrosis.

Not surprisingly, given the degree of lung fibrosis induced bybleomycin, animals treated with this agent had reduced oxygen content intheir blood, and lost weight over the course of the two weeks ofobservation. Both symptoms of poor lung function were normalized by theSAP treatment. Such secondary effects provide convenient measures ofutility that can be measured non-invasively and are thus useful indefining a clinical profile of SAP as a therapeutic agent.

Although only exemplary embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of these examples are possible without departing from thespirit and intended scope of the invention.

1. A method of suppressing fibrocyte formation in a mammal sufferingfrom kidney fibrosis, the method comprising administering to a mammal inneed thereof a composition comprising Serum Amyloid P (SAP) protein or aportion thereof that binds to Fcγ receptors (FcγR) and inhibitsfibrocyte differentiation from monocytes in an amount sufficient tosuppress kidney fibrosis.
 2. The method of claim 1, wherein SAP or aportion thereof has a concentration in the composition of at leastapproximately 0.5 μg/ml.
 3. The method of claim 1, wherein administeringthe composition comprises administering by intravenous injection.
 4. Themethod of claim 1, wherein the mammal is a human.
 5. The method of claim1, wherein said SAP or a portion thereof is conjugated to abiocompatible polymer.
 6. The method of claim 5, wherein thebiocompatible polymer is selected from PEG, a poly(amino acid), and apolysaccharide; or copolymers and combinations thereof.
 7. The method ofclaim 1, further comprising administering a composition selected fromIL-12, Laminin-1, IgG aggregates, cross-linked IgG and combinationsthereof.
 8. The method of claim 1, wherein administering comprisesadministering approximately 1.6 μg SAP or a portion thereof per gram ofmammal.
 9. The method of claim 1, wherein administering comprisesadministering an amount of SAP or a portion thereof sufficient toapproximately double the normal serum concentration of SAP or a portionthereof similar to the portion thereof administered in the mammal. 10.The method of claim 1, wherein administering comprises administering anamount of SAP sufficient to increases the normal serum concentration ofSAP or a portion thereof similar to the portion thereof administered inthe mammal by approximately 25%.
 11. The method of claim 1, wherein theSAP protein or a portion thereof forms a pentamer.
 12. The method ofclaim 1, wherein the SAP protein or a portion thereof includes one ormore sequences selected from: KERVGEYSLYIGRHKVTSKVIEKFP (SEQ. ID. NO.1), NILSAYQGTPLPA (SEQ ID NO: 2) and IRGYVIIKPLV (SEQ ID NO: 3).
 13. Themethod of claim 1, wherein administering comprises administeringapproximately 600 μg or more of the SAP protein or a portion thereof.14. The method of claim 1, wherein kidney fibrosis comprises a conditionselected from sclerosing glomerulonephritis, nephrogenic fibrosingdermopathy, diabetic nephropathy, renal tubulointerstitial fibrosis, andfocal segmental glomerulosclerosis.
 15. A method of suppressingfibrocyte formation, comprising administering to a patient an agent thatactivates intracellular signaling in monocytes consistent with FcγRactivation by SAP, in an amount and for a time sufficient to suppressdifferentiation of the monocytes into fibrocytes.
 16. A method oftreating a fibrotic or fibroproliferative disorder in a patient, themethod comprising administering to a patient suffering from or at riskof developing said disorder an agent that induces intracellularsignaling in monocytes consistent with FcγR activation by SAP, in anamount and for a time sufficient to suppress differentiation of themonocytes into fibrocytes.
 17. A pharmaceutical preparation forsuppressing fibrocyte formation in a patient, comprising an agent thatactivates intracellular signaling in monocytes in a manner consistentwith FcγR activation by SAP, suitable for administration to a human orveterinarian patient.
 18. A method for treating asthma, comprisingadministering SAP or SAP agonist.
 19. A method for reducing remodelingof a patient's airways in response to inflammation, comprisingadministering to a patient an agent that activates intracellularsignaling in monocytes in a manner consistent with FCγR activation bySAP, in an amount and for a time sufficient to reduce one or moredifferentiation of monocytes into fibrocytes, differentiation offibrocytes into myofibroblasts, and fibrosis.
 20. A method for treatinga pulmonary disease involving differentiation of monocytes intofibrocytes, comprising administering to a patient an agent thatactivates intracellular signaling in monocytes in a manner consistentwith FcγR activation by SAP, in an amount and for a time sufficient toreduce said differentiation of monocytes into fibrocytes.
 21. Apharmaceutical preparation for inhalation by a patient, comprising anagent that activates intracellular signaling in monocytes in a mannerconsistent with FcγR activation by SAP, suitable for administration to ahuman patient.